Aberrant methylation of CpG islands in the p16 (CDKN2A) gene can be used for early prediction of malignant transformation of epithelial dysplasia, a precancerous lesion (Sun et al. Clin Cancer Res 2004, 10:5087-5093; Cao et al. Clin Cancer Res 2009, 15:5178-5183). The related artificial nucleotide sequences used in these studies and their uses were granted the patent rights in China and in Europe (Chinese Patent No. ZL03826140.5: European Patent No. EP1602631).
In these studies, the methylation is detected using the method including following steps: sodium bisulfite modification of unmethylated cytosines in the CpG island; after the sodium bisulfite modification, the sequence of sense-strand DNA is no longer complementary to the antisense-strand DNA, and the double-stranded DNA becomes 2 single-stranded DNAs. The sodium bisulfite-modified single-strand antisense DNA is a good template for designing amplification primers used in a methylation-specific PCR assay (150 bp p16-MSP; Herman et al PNAS 1996, 93:9821-9826) and quantitative methylation-specific 70 bp fluorescence assays (MethyLight; Ead et al. Cancer Res 2000, 60:5021-5026), which are commonly used in scientific researches.
In our prospective epidemiological follow-up cohort study, we have found that methylated p16 (p16M) detected with the 150 bp-MSP method (150 bp p16-MSP; Herman et al PNAS 1996, 93:9821-9826) can be used to predict malignant transformation of oral mucosal epithelium. The sensitivity and specificity of prediction in the elderly over 60 years old are 76.9% and 78.3%, respectively (Cao et al, Clin Cancer Res 2009, 15:5178-5183). These results show that these CpG sites in the p16 genes may have important application value.
As a qualitative assay, the 150 bp-MSP assay can't meet with the requirements for developing diagnostic kit, because said diagnostic kit need to use the sequence-specific probes as to confirm and make the quantitative determination. If the results of 150 bp-MSP analysis are used as the gold standard, we have found that the qualitative results (without setting of a cut-off value) of the 70 bp-MethyLight fluorescence analysis (MethyLight; Ead et al. Cancer Res 2000, 60:5021-5026) has a higher sensitivity (37/44=84.1%), poor specificity (20/58=34.5%), and low accuracy (84.1%+34.5%−1=18.6%), as shown in FIG. 1. Even though the employment of a cut-off value in the MethyLight analysis can improve its accuracy, statistically significant correlation still cannot be observed between the MethyLight determination and clinical outcomes. Apparently, the 70 bp-MethyLight fluorescence method can not meet the requirements of the development of diagnostic kits.